This invention relates to an improved polychromatic spectrophotometer method and apparatus which is particularly useful in the field of liquid chromatography in which a solvent solution carrying one or more materials to be analyzed is introduced into a chromatograph column, and the eluent issuing from the column is optically analyzed in a sample cell.
Chromatography has proved to be an extremely useful tool in both research and routine testing to provide rapid high accuracy chemical analysis. In a typical chromatographic procedure, the material whose composition is to be tested is dissolved in a solution consisting of one or more solvents, and is then pumped through a chromatographic column containing constituents such as silica or resins which cause different constituents of the material being tested to traverse the column at different predictable rates. The result is that different optical manifestations corresponding to the presence of different constituents are available for observation at different instants of time as the eluent issues from the chromatograph column.
One of the useful methods of obtaining readings from the chromatograph is by the use of a spectrophotometric detector which provides a chart of sample component separation in terms of radiation absorbance at what is essentially a single selected wavelength of radiation with which the sample is illuminated in a sample cell at the end of the chromatographic column. Stated in another way, the sample may be illuminated with a relatively broad spectrum of illumination (say in the ultraviolet region) and the radiation absorbance is recorded at only one wavelength, such as for instance at 255 nanometers. An absorbance versus time plot of this nature is illustrated in FIG. 1. In that figure, the various peaks signify the presence of various different chemical constituents which are detectable at the particular selected illumination wavelength, the height of each peak being a function of the concentration of the constituent represented by the peak and the degree of absorbance of illumination caused by the manifestation of that particular constituent.
Another extremely valuable output from a spectrophotometric detector used with a chromatograph is an output of the nature illustrated in FIG. 2 of the accompanying drawings which represents the absorbance vs wavelength when taken at a particular instant of time after initiation of the chromatographic separation. This is referred to as a spectral plot or spectrum. Until very recently, it was not possible to obtain such a plot except by stopping the flow of fluid through the column, mechanically scanning a monochrometer, and repeating the same cycle over and over, generating a different spectrum on each cycle. The problem with this procedure is that it is very time consuming, and accuracy may be compromised by minor variations in conditions from one test to another.
In recent years, so-called charge-coupled diode array spectrophotometric detectors have been used in liquid chromatography which attempt to produce the spectral plot corresponding to that of FIG. 2 in a single pass of the fluid through the liquid chromatography column, and without stopping the flow of fluid eluent. Such arrangements are described, for instance, in an article by Stuart A. Borman entitled "Charge-coupled Diode Array Detectors for LC" which appeared on pages 836A through 842A of "Analytical Chemistry" volume 55, number 8, July 1983.
Such charge-coupled diode array systems for liquid chromatography are attractive for what they attempt to do, but they suffer from a number of very serious problems and deficiencies. For instance, they are quite expensive and complicated in structure, slow in operation, offer a low signal-to-noise ratio, and a low accuracy, and a limited dynamic range. The charge-coupled diode arrays themselves are very expensive. The diodes in the array are back-biased so that they operate as capacitors in parallel with photoresistors. In some arrays, additional capacitors are built into the array.
Typically, in operation, all of the diode capacitors are charged to a uniform voltage level, and then discharged on the basis of illumination received, the charge-coupled diodes operating like photoresistors. The degree of discharge is thus supposed to be a measure of the amount of illumination received, and the degree of discharge is measured each time the device is strobed to recharge the capacitor. One very serious source of error is that it is very difficult to repeatedly recharge the devices to exactly the same level. Also, unfortunately, the devices have an appreciable dark discharge current which is subject to substantial change, particularly in response to changes in temperature. Accordingly, it is necessary to constantly recalibrate the diodes for dark discharge current by using mechanical shutters which darken the diodes, strobing all of the diodes to derive dark discharge values, storing those values, and then subtracting those dark discharge values from the subsequent readings during illumination. This limits accuracy and also creates a tremendous overhead in terms of digital storage space and processing time. It also slows down the total process and limits the number of scans available during a particular interval of time.
Thus, while the measurement scan is claimed to be as short as 1/100th of a second, the fastest claimed repeated sampling rate is only 25 times per second. Another major factor in reducing the overall speed of the charge-coupled diode system and increasing the overhead in terms of digital storage space and processing time is that the system is so lacking in accuracy that the results of four scans are taken and the individual readings of the four scans are averaged to provide a higher accuracy result. This accounts for the fact that the scan may take only 1/100th of a second, but the fastest sampling rate is only 25 times per second. Furthermore, the strobing of the devices in the charge-coupled diode array systems creates substantial "noise" (undesired signals) substantially reducing the so-called signal-to-noise ratio (the ratio of useful signals to noise signals).
The dynamic range of the devices is inherently limited on the upper end by the fact that there is only a certain amount of charge which can be discharged from each device in response to a strong optical signal. Unfortunately, the amount of charge which can be discharged from each device is seriously limited by the fact that the operation very soon gets into a nonlinear range as discharge proceeds. The range and accuracy are also limited on the low side because it is difficult to accurately measure a very small optical signal based upon the measurement of a small difference between two large quantities. Thus, the measurement involves measuring a difference between the total charge as reduced to a very minor extent by a small optical signal, and the subsequent recharge value.
The accuracy is further seriously limited by the fact that the acquisition of the data is carried out in a scan cycle which is too slow to capture the data for all of the spectral segments required for the spectral plot of the entire scan spectrum before the sample concentration in the sample cell changes appreciably because of the flow of eluent through the cell. If this source of inaccuracy is to be reduced, the eluent flow rate must be reduced, thus substantially reducing the efficiency of the system. Similarly, the rate at which successive total spectrum samples can be taken is typically a maximum of 25 per second, a rate which also may require a reduction in the eluent flow rate and a consequent reduction in efficiency of the system.
The above-mentioned Analytical Chemistry article indicates a clear recognition of many of the above-mentioned problems, including the high cost of the photodetector array, and the fact that the prior single wavelength and variable wavelength liquid chromatograph detectors are cheaper and inherently more sensitive than the charge-coupled diode detector array systems.
While the article mentions "simultaneously monitoring all wavelengths in the spectrum", it does not really operate "simultaneously" but, as discussed above, in a scan which takes at least 1/100th of a second, and typically more time up to 21/2 seconds.